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integrating microcoils for trapping magnetic nano

particles for biological applications

Hong Ha Cao

To cite this version:

Hong Ha Cao. The fabrication process of microfluidic devices integrating microcoils for trapping magnetic nano particles for biological applications. Other [condmat.other]. Université Paris Sud -Paris XI, 2015. English. �NNT : 2015PA112150�. �tel-01350864�

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UNIVERSITÉ PARIS SUD

L’Institut d’Électronique Fondamentale

Ecole doctorale: Sciences et Technologies de l’Information des Télécommunications et des Systèmes (STITS – ED 422)

Discipline: physique

Microsystèmes pour Biomédical

Thèse de doctorat

Présentée par :

CAO Hong Ha

Sujet: Procédé de fabrication de dispositifs microfluidiques intégrant des microbobines – Piégeage de nanoparticules magnétiques pour des applications en

biologie

Composition du jury:

Mme Isabelle MABILLE MCF, HDR Rapporteur Université Pierre et Marie Curie Institut de Recherche de Chimie Mr Benoit PIRO PR Rapporteur Université Paris Diderot

Laboratoire ITODYS

Mr Bruno LEPIOUFLE PR Examinateur Ecole Normale Supérieure de Cachan SATIE

Mme Claire SMADJA PR Examinateur Université Paris Sud Institut Galien paris-Sud Mme Marion WOYTASIK MCF Examinateur Université Paris-Sud

Institut d'Electronique Fondamentale Mme Elisabeth DUFOUR-GERGAM PR Directeur de thèse Université Paris-Sud

Institut d'Electronique Fondamentale Mr Emile MARTINCIC MCF Invité Université Paris-Sud

Institut d'Electronique Fondamentale Mr Mehdi AMMAR MCF Invité Université Paris-Sud

Institut d'Electronique Fondamentale

July 21

st

, 2015

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For my family,

With the deep gratitude for the encouragement to overcome the hard time when I studied in France and had to live far from you for over three years from March 2012

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i | P a g e Acknowledgment

Foremost, I would like to express much appreciation to my supervisor Prof. Elisabeth DUFOUR-GERGAM, who gave me a chance to be a PhD. student at her group (MicroSystèmes pour le Biomédical, L’Institut d’Électronique Fondamentale (IEF), l’Université Paris Sud) in the first time I met her in Hanoi, Vietnam over past three years. She helped me a lot to make plan and scientific ideal for my research in the topic of microfluidic devices and application, and she created best condition for me to approach environment working with modern machines in clean room of laboratory. I also sincerely thank her financial support for my experiment and life during the time I studied at IEF. In addition, she also wholeheartedly helped me a lot in administrative formalities of university and laboratory, specially in documents to the Ministry of Education and Training – Vietnam. One more time, I would like thank for her help in facilitating a good relationship with other colleagues in past recent time.

I would like to give a precious thanks to Dr. Marion WOYTASIK, who helped directly me a lot as a supervisor. Although she was very busy, she had carefully guided me from first technics of basic and simple experiments when I began to approach the topic of my research. In addition, she also helped thoroughly me to overcome difficulties in writing, correcting papers and manuscripts, as well as in matters of documents in French.

I also would like to give a sincere thanks to Dr. Emile MARTINCIC and Dr. Mehdi AMMAR, who spent much time to help me in the theory, simulation and practice of the magnetic field in chapter 3 and papers, the theory and applications of biology in chapter 4. They gave me guidance in detail in designing microcoils, performing experiments with trapping magnetic beads and bead-based immunoassays. They also carefully helped me to write and correct my thesis. All the assessments, comments and correction in my writing improved my skin in writing and specialist knowledge of research objectives.

I would like to thank Assc. Prof. TRAN Dai Lam in Institute of Materials Science, Vietnam Academy of Science and Technology, who introduced me to Prof. Elisabeth DUFOUR-GERGAM and then I had opportunity to be a PhD. student in IEF, University Paris Sud. He also gave me useful discussion in my topic research.

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I would like to sincerely thank Prof. Alain BOSSEBOEUF, who supported me to get an opportunity to participate the Namis School September, 2013 in Korea. He also supported me documents and procedures involving in the administration of laboratory. With supports of him and Prof. Elie LEFEUVRE about the equipment helped me to obtain results in this thesis. Kindly thanks gives to Dr. Johan MOULIN, who helped me in the beginning of the magnetic calculation and equipment setup for the first experiment of trapping magnetic beads. I would like to give a special thanks to Dr. Philipe COSTE, who helped me a lot in problems involving to information technology and computers, and also to Prof. Pierre-Yves JOUBERT, who helped me to apply all documents to Laboratory IEF, thus, I had a chance to have financial support from Laboratory. I also sincerely thank Ms. Magdalèna COUTY, who enthusiastically helped me to carry out first experiments in a clean room of IEF in fabricating PDMS microfluidics, she also helped me about documents in French during the time I worked with her office No. 102 Building 220, IEF. I would like to thank Ms. Meritxell CORTES, who helped me a lot in the fabrication of microcoils and English writing, we had friendly time when she worked at IEF.

I sincerely thank Prof. Niko HILDEBRANDT, who gave me the best working conditions when I worked with the fluorescence microscopy for experiments. I also thank colleagues as Shashi, Stina... in Bio Nano Photonics team, who guided me using the fluorescence microscopy. A sincere thank give to Dr. Claire SMADJA, who helped and supported me to take first ELISA experiments in the laboratory of Paris-Sud Institut Galien, Faculté de Pharmacie, Université Paris-Paris-Sud. In this ELISA experiments, I also would like to thank Kiarach Mesbah, GIANG Phuong Ly, Mohamed MIRAOUI, who helped me to obtain first experiences about working in biology.

I would like to give my sincere thanks to my colleagues DINH Thi Hong Nhung, Magdalena COUTY, Feriel HAMDI, who worked with me in project of reversible packaging of PDMS microfluidic chip. I would like to thank Monica ARAYA-FARIAS, who worked with me on PDMS microchip fabrication, and LE VAN Quynh, who helped me in the SEM measurement.

I would like to sincerely thank all my Vietnamese friends, who have friendly shared in works and life during the time I worked and stayed in France. Those of my friends helped me a lot in the administrative procedure in French: ĐINH Thị Hồng

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iii | P a g e Nhung, Vũ Thị Nhung, MAI Văn Huy, LÊ Thanh Nghị. Other friends (Hưng, Trường, Cẩm, Chung, Lâm, Vân Anh, Hoàng, Quỳnh, Mai, Trang, Huy, Tuấn, Phương, Khuyến, Tiệp+Nương…) sincerely shared with me many things in works and life. I am also happy to have French friends (Aida, Sarah, Vincent, Alexandre, Guillaume, Iman, Pierre,…).

Most importantly and above all, I would like to give my love and thank to my family, specially to my wife, NGUYỄN Vân Anh, who spent much time to take care with love my two daughters, CAO Hải Anh and CAO Phương Linh, although she was

very busy and her health was not good. They are always a great motivation to encourage me during the time I worked in France and I had to live away from home.

I would like to express my great gratitude to my parents, my parents-in-law, who encouraged and helped me and my wife and children in over three years ago. I also express my most sincere thanks to all big family members, who encouraged me in finishing this thesis.

I would like to thank the French RENATECH network and the Vietnamese Overseas Scholarship Program (Project - 322) of the Vietnamese government for the financial support during the study period at IEF, Univ. Paris-Sud, France. I also thank colleagues at Department of Physical chemistry, School of Chemical Engineering, Hanoi University of Science and Technology, Vietnam, who facilitated my study in France.

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v | P a g e CONTENTS Acknowledgments ... i Résumé/Summary ... ix Nomenclature ... x CHAPTER 1. CONTEXT ... 1

CHAPTER 2. REVIEW OF MICROFLUIDICS FOR BIOLOGICAL APPLICATIONS ... 5

2.1 Short overview of the historical development of microfluidics ... 6

2.2 Introduction to microfluidic system ... 7

2.2.1 Microfluidics concepts ... 7

a. Generalities ... 7

b. Advantages of microfluidics in chemical and biological applications: ... 9

2.2.2 Microfluidics’ theoretical aspects ... 10

a. Reynolds number ... 10

b. The profile of velocity flow in a rectangle channel ... 11

2.3 Magnetic bead-based immunoassay ... 11

2.3.1 Magnetic micro/nano beads in immunoassay ... 12

a. Introduction of functionalized magnetic beads ... 12

b. Magnetic properties of the materials of bead’s core ... 14

2.3.2 Immunoassay principles ... 15

2.3.2.1 Introduction of immunoassay ... 15

2.3.2.2 Bio-components in immunoassay ... 16

a. Capture antibody and immuno-sorbent/solid phase ... 17

b. Detection antibodies (antibodies conjugated enzyme) ... 19

c. Enzyme and antigens ... 20

2.3.3 General protocol of magnetic bead-based immunoassay ... 20

2.4 Microfluidics in magnetic bead-based immunoassay ... 22

2.4.1 Magnetism and trapping/transporting magnetic beads in microfluidics ... 22

2.4.1.1 Permanent magnet for manipulating magnetic beads in microfluidics ... 22

2.4.1.2 Electromagnet for manipulating magnetic beads in microfluidics ... 24

2.4.2 Bead-based immunoassay inside microfluidics ... 28

2.4.3 Microfluidics fabrication ... 30

2.4.3.1 Microchannel fabrication ... 30

a. Materials for microchannel network ... 30

b. PDMS as a popular materials for microfluidics ... 32

2.4.3.2 Packaging PDMS microfluidic chip ... 33

a. Irreversible bonding ... 33

b. Reversible bonding ... 34

2.5 Conclusions ... 36

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CHAPTER 3. MICROCOILS DESIGN AND FABRICATION ... 41

3.1. Introduction ... 42

3.2. Magnetic field/force theory ... 43

3.2.1. Magnetic field of straight and finite wire ... 43

3.2.2. Magnetic force: theory ... 47

3.3. Magnetic field simulation and calculation ... 48

3.3.1. FE simulation of microcoils ... 48

3.3.2. Simulation results... 50

a. General considerations on the magnitude and shape of the magnetic field ... 50

b. Coil geometry effect on the power loss and heating ... 53

c. Power consumption and merit factors ... 55

d. Conclusions: ... 57

3.4. Planar coils fabrication and characterization ... 57

3.4.1. Mask design ... 58

3.4.2. Fabrication process of coils ... 59

3.5. Conclusions ... 62

References ... 63

CHAPTER 4. FABRICATION OF MICROFLUIDIC CHIPS AND APPLICATIONS ... 65

4.1. Introduction ... 66

4.2. Microfluidic chips fabrication ... 67

4.2.1. PDMS channel ... 67

a. Design of PDMS channel network ... 67

b. Channel fabrication. ... 68

4.2.2. Valves switch ... 70

a. Design of multi-valves switch ... 71

b. Multi-valve switch fabrication... 72

4.2.3. Microfluidic chip packaging ... 73

4.2.3.1. PDMS / PDMS reversible packaging based on stamping technique ... 74

4.2.3.2. Reversible packaging based on anti-adhesive under-layer ... 75

4.2.4. General conclusions in microfluidic chip fabrication: ... 77

4.3. First trapping experiments with microfluidic chip ... 79

4.3.1. Study on the temperature of the microchip ... 79

a. Experiment description ... 79

b. Results and discussion... 80

4.3.2. Magnetic nanobeads trapping experiments ... 81

a. Magnetic beads ... 81

b. Microfluidic chip ... 82

c. Experiment description ... 83

4.3.3. Results and discussion ... 85

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b. Trapping magnetic beads in a wide channel: current value effect ... 86

c. Trapping magnetic beads in a wide channel: time cumulative experiment ... 88

4.3.4. Conclusion of trapping experiment ... 90

4.4. Microfluidic chip for on-chip magnetic beads-based immunoassay ... 91

4.4.1. Modification of PDMS surface ... 91

4.4.1.1. Materials and surface modification protocol ... 92

a. Materials: ... 92

b. Protocol of surface modification and bonding process ... 93

4.4.1.2. Results and discussions ... 95

a. The effect of PEO treatment on PDMS surface ... 95

b. The effect of BSA and PEO treatment on PDMS surface ... 96

c. Roughness study by AFM ... 97

d. Adsorption of fluorescent dye on modified PDMS surface ... 98

4.4.2. First steps in bead-based immunoassay inside microfluidic chip ... 100

4.4.2.1. General IgG grafting protocol ... 101

a. The preparation of materials ... 101

b. IgG grafting protocol ... 103

4.4.2.2. Experiment and results in micro test tube (control experiment) ... 104

a. Protocol description ... 104

b. Results and discussion ... 105

4.4.2.3. Experiments in microfluidic chip ... 108

a. Setup of microfluidic chip for grafting IgGs: ... 109

b. IgG grafting protocol ... 110

c. Results and discussion ... 113

4.4.3. Conclusions from applying microfluidic chip with embedded microcoil in immunoassay . 118 4.5. General conclusion of chapter 4 ... 119

References ... 120

5. Conclusion and prospects ... 123

APPENDICES ... 127

Publications: ... 128

APPENDIX 1. Chapter 3 – Microcoils design and fabrication ... 129

APPENDIX 2. Paper “Reversible bonding by dimethyl-methylphenylmethoxy siloxane – based stamping technique for reusable poly(dimethylsiloxane) microfluidic chip” ... 134

APPENDIX 3. Chapter 4 - Leakage test and peeling off channel ... 139

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Résumé

Le but de cette étude est de concevoir, fabriquer et caractériser une puce microfluidique afin de mettre en oeuve la capture de nanoparticules magnétiques fonctionnalisées en vue de la reconnaissance d’anticorps spécifiques (couplage d’une très grande spécificité et sensibilité). Après avoir modélisé et simulé les performances de la microbobine intégrée dans le canal de la puce microfluidique en prenant soin de limiter la température du fluide à 37°C, la capture devant être effective, le microsystème est fabriqué en salle blanche en utilisant des procédés de fabrication collective. La fabrication du microdispositif en PDMS a aussi donné lieu à l’optimisation de procédés de modification de surface afin d’assurer la ré-utilisation du microdispositif (packaging réversible) et la limitation de l’adsorption non spécifique.

L’immobilisation des anticorps su les billes (300 nm) a été menée à l’intérieur du canal en utilisant un protocole de type ELISA éprouvé. Le procédé a montré qu’il était également efficient pour cet environnement puisque nous avons pu mettre ne évidence la capture de nanoparticules.

Summary

In this study, a concept of microfluidic chip with embedded planar coils is designed and fabricated for the aim of trapping effectively functionalized magnetic nanobeads and immobilizing antibody (IgG type). The planar coils as a heart of microfluidic chip is designed with criterion parameters which are optimized from simulation parameters of the maximum magnetic field, low power consumption and high power efficiency by FE method. The characterization of microcoils such as effectively nanobeads (300 nm) at low temperature (<37oC) is performed and confirmed. The channel network in PDMS material is designed for

matching with entire process (including mixing and trapping beads) in microfluidic chip. A process of PDMS’s surface modification is also carried out in the assemble step of chip in order to limit the non-specific adsorption of many bio substances on PDMS surface. The microfluidic chip assemble is performed by using some developed techniques of reversible packaging PDMS microfluidic chip (such as stamping technique, using non-adhesive layer, oxygen plasma combining with solvent treatment). These packaging methods are important to reused microchip (specially the bottom substrate) in many times.

The immobilization of antibody IgG-type is performed inside microfluidic chip following the standard protocol of bead-based ELISA in micro test tube. The result showed that IgG antibodies are well grafted on the surface of carboxyl-beads (comparing to result of standard protocol); these grafted antibodies are confirmed by coupling them with labeled second antibody (Fab-FITC conjugation).

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Nomenclature

Abbreviation Acronyms

Ab, Abs Antibody, antibodies

Ag, Ags Antigen, antigens

BSA Bovine serum albumin

CMOS Complementary metal–oxide–semiconductor

DMPMS Dimethyl methylphenylmethoxy siloxane

ELISA Enzyme-Linked ImmunoSorbent Assay

FEM Finite element method/modelling

FITC Fluorescein isothiocyanate

HRP horseradish peroxidase

IBE Ion beam etching

IgG Immunoglobulin G

Igs Immunoglobulins

LoC lab-on-a-chip

MB/MBs Magnetic nano bead/beads

MEMS/NEMS Micro/Nano electronic mechanic systems

mNP/mNPs Magnetic nano particle/particles

NP/NPs Nano particle/particles

PBS Phosphate buffered saline

PDMS Poly(dimethyl siloxane)

PECVD Plasma-enhanced chemical vapor deposition

PEO Poly(ethylene oxide)

RIE Reactive-ion etching

SU-8 Epoxy based negative photoresist

TAS/μ-TAS Total analysis system / micro total analysis system

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CHAPTER 1. CONTEXT

Microfluidics: in search of a killer application

“Companies and academic researchers are developing more and more microfluidic devices. But what the technology stakeholders really want is an application that will trigger widespread adoption of microfluidics by biologists”. Nathan Blow reported in [1].

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In analytic biochemistry assay, to specifically detect the presence of a target/substance (an antigen) in analyte, the Enzyme-Linked Immune-Sorbent Assay (ELISA) has been used widely as a diagnostic tool in medicine, biology, and plant pathology. This nowadays well known method was developed based on the radioimmunoassay, which is a technique using radioactively labeled antigens or antibodies and was first published in 1960 by Rosalyn Sussman Yalow and Solomon Berson [2]. Until the 70s of the 20th century, the ELISA method was developed and became one of the most specific method in immunoassay (i.e. HIV virus, Alzheimer or cancerous biomarker [3-6]), a keyword in literature [7-10]. Basically, ELISA is typically based on the “sandwich” structure of primary antibody (Ab) – antigen (Ag) – second antibody (with labeled conjugation). The detection of Ab or Ag, is performed easily in two ways: direct ELISA and indirect ELISA (including supported surface or capture antibody as basic platform), by using fluorogenic, electrochemiluminescent, this method presents various advantages like high sensitivities/ultrasensitive and the possibility to multiplex the detection [11].

Figure 1.1. An example of Common ELISA formats. In the assay, the antigen of interest is immobilized by direct adsorption to the assay plate or by first attaching a capture antibody to the plate surface. Detection of the antigen can then be performed using an enzyme-conjugated (E) primary antibody (direct detection) or a

matched set of unlabeled primary and conjugated secondary antibodies (indirect detection)(1).

Figure 1.1 shows the three common ELISA formats. In the direct detection format, the capture antibody that reacts directly with the antigen is a labeled primary antibody. Direct detection can be performed with antigen that is directly immobilized on the assay plate or with the capture assay format. Direct detection is not widely used in ELISA but is quite common for immuno-histochemical staining of tissues and cells. Secondly, the indirect detection method uses a labeled secondary antibody for detection. The secondary antibody has specificity for only the primary antibody (i.e. capture antibody) or the assay will not be specific for the antigen. Generally, this is achieved by using capture and primary antibodies from different host species (e.g.,

(1) https://www.lifetechnologies.com

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mouse IgG and rabbit IgG, respectively). For sandwich assays, it is beneficial to use secondary antibodies that have been cross-adsorbed to remove any antibodies that have affinity for the capture antibody.

Traditional ELISA tests are performed in wells, which are passively bind antibodies and proteins. Recent evolutions of the technic involving nanobeads present a lot of advantages for future analysis applications. The use of functionalized magnetic nano/micro beads allows the detection of ultralow concentration (few zepto-grams) of targets (proteins, cells) in extremely small volume (few μL) of analyte [6, 12-15]. Currently, the on-chip bead-based ELISA system (“snapshot” ELISA techniques) has higher sensitivity than standard ELISA techniques, using magnetic micro/nano beads to increase surface-to-volume ratio combined with microfluidic devices using major bead magnetic trapping methods (by permanent magnet [16, 17] or electromagnet [13]).

Let us recall that many typical microfluidic devices are currently under development, ranging from single components such as flow sensors and valves for gas pressure regulation, to complex micro-fluid handling systems for chemical and biological analyses. Many function modules (such as sensors) can be integrated on a single substrate or complex modules of microfluidics. Specially, researchers had an ambition to make these devices portably, or to integrate many components into hand-held format for variety of point-of-care application.

In this study, we propose to develop a new type on-chip bead-based immunoassay platform with embedded planar coils, designed and fabricated for the aim of trapping effectively functionalized magnetic nano/micro beads, and the immobilization of capture antibodies inside microchannel.

In immunoassay procedure, the planar coils will play an important role of separating/sorting magnetic beads.

The study will be presented in four chapters describing first some items about microfluidics and immunoassays bottlenecks, the simulation of the microcoils to optimize the device, the fabrication and the process development used for the chip fabrication, the first biological experiments and, at last the conclusion and emerging ideas.

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We will see in the chapter 3 the microcoils designed with criterion parameters optimized from simulation parameters to obtain maximum magnetic field, low power consumption and high power efficiency. These values are calculated by Finite Element (FE) method thanks to ANSYS® software. Simulation results will also predict the affection of coil’s geometries (including the size, number of turn, parameters of conductors, etc.) on power loss and heating of coils while they are working. We will also study the surface temperature during operation which is so important to take into account for biological medium. This chapter will finish with the microcoils fabrication process. In the chapter 4, we will present some original processes to assemble the bottom substrate (holding the microcoils) with PDMS cap holding the microchannel network. Three techniques of reversible bonding are developed: (i). Coating layer of non-adhesive material; (ii). Stamping technique using dimethyl-methylphenylmethoxy siloxane (DMPMS) conformal coating as adhesive layer; (iii). Oxygen plasma treatment for bonding PDMS to PDMS. We also studied the process to functionalize the beads outside and inside the chip and the trapping of the specific elements.

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CHAPTER 2. REVIEW OF MICROFLUIDICS FOR

BIOLOGICAL APPLICATIONS

- Introduction of microfluidic systems - Review of historical development

- Introduction of processes and components in microfluidic chip

“Microfluidics, a technology characterized by the engineered manipulation of fluids at the submillimeter scale, has shown considerable promise for improving diagnostics and biology research. Certain properties of microfluidic technologies, such as rapid sample processing and the precise control of fluids in an assay, have made them attractive candidates to replace traditional experimental approaches.” Eric K.

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2.1 Short overview of the historical development of microfluidics

Microfluidics attracts the attention of scientists since the1950s who desire to manipulate small volume (micro/nano litter) of liquids in micro channels/capillaries. It is marked by revolution in exploiting molecular distributions between mobile and stationary phase in a small column [19] and capillary electrophoresis. It was known that, processes in very small and long capillary had some advance performances thanks to the large surface-to-volume ratio. Until 1970s, some first microfluidic devices were used in the research of gas chromatograph at Stanford University [20] and ink jet printer nozzles on silicon wafer at IBM [21, 22]. In the early 1990s, Manz et al. [23] reported a microfluidic potential for addressing issues facing analytical methods, now it is called micro Total Analysis System (µTAS), and hence the term of “Lab-on-a-chip” – LoC was introduced. It can be said that microfluidics has been become “hot” research topic in chemical and biological analysis since then [18]. From the 2000s, microfluidics has the potential to significantly change the way modern biology is performed, [24]. In many conventional assays, it’s possible to integrate many analytical steps (such as sample loading, rinsing, reaction, separation, mixing, detection, etc.) into single or fully-automated platform. By these advantages, the Lab-on-a-chip system can greatly reduce the cost and time in each analysis and multiplexed analysis can be developed.

Nowadays, many different microfluidic devices ranging from single components such as flow sensors and valves for gas pressure regulation, to complex micro-fluid handling systems for chemical and biological analyses can be integrated on a single substrate. The dream of the scientists of the 1970s researchers seams very near because it is now possible to make portably devices integrating many components into hand-held format for variety of point-of-care (PoC) application. It can take fast or mobility analysis.

In chemical, medical and biological analysis, microfluidics can be used in some major applications: measuring molecular diffusion coefficients [25]; pH [26], chemical binding coefficients [27], and enzyme reaction kinetics [28]. Other applications for microfluidic devices include capillary electrophoresis [29], immunoassays [30], flow cytometry [31], sample injection of proteins for analysis via mass spectrometry [32], PCR amplification [33], DNA analysis [34], cell analysis [15] and chemical gradient

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formation [35], clinical diagnostics [30, 36]. We will see very shortly some of them in the next sections.

2.2 Introduction to microfluidic system 2.2.1 Microfluidics concepts

a. Generalities

Microfluidic chip can be identified as a micro device that deals with the flow of liquid inside one or more tiny channels with at least one dimension of nano/micrometers size. Concerning the terms of microfluidics, it can be considered to both science and technology (including: research on theoretical of flows, transport phenomena, interaction in tiny space with the high surface area to volume ratio; microfabrication technology of microfluidic chip for application in chemical/biological analysis – such as Lab-on-a-chip). An example of microfluidic devices is shown in Figure 2.1 (a) (1). This microfluidic chip is used as a micro-reactor and micro-mixture device of two substances injected in inlet 1 and 2. The reaction’s product is released through outlet.

Inlet 1

Inlet 2

Outlet

(a) (b)

Figure 2.1. (a) Microfluidic chip using as micro-reactor and micro-mixture device (1); (b) A high-throughput

automated cell culture system with integrated multiplexer, peristaltic pump, cell inlet and waste output, [37].

Another example is shown in Figure 2.1 (b), a complicated microfluidic chip with multi-inlet and outlet is used as a high-throughput automated cell culture system with integrated multiplexer, peristaltic pump, cell inlet and waste output [37, 38].

Regarding the miniaturization of the fluidic devices, many researchers realized the benefits of working in small scale of microfluidic devices, because the process inside microchannel has advantages of new effects and better performance. Commonly, the scale of microfluidics corresponds to the scale of study’s objectives. The relationship between the volume scale of analyte and length scale of size’s devices is shown in

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Figure 2.2. It also shows the size characteristics of typical microfluidic devices compared to other common objects.

Figure 2.2. Size characteristics of microfluidic devices [39].

 Microfluidics chip is commonly structured from basic modules:

- Channel network (including parts of driving flows, function parts for mixing, separating flow, creating droplets or trapping, etc.). Figure 2.3 shows an example of microfluidic chemotaxis device [40]. The device consists of a gradient-mixing module and a chemotaxis observation module. This device is developed for generating precise and stable gradients of signaling molecules to investigate the effects of individual and combined chemo-effector gradients on Escherichia coli chemotaxis.

Figure 2.3. Microfluidic chemotaxis device. (A) Schematic representation of the microfluidic device. (B) Food dye representation of gradient formation in the microfluidic device, showing the formation of a range of

greens from blue and yellow inputs.

- Bottom substrate contains integrated analyzing components (i.e. electrochemical sensors, trapping particles (including magnetic or non-magnetic particles, biomolecules or cluster of molecules, etc.), temperature sensors, heater, optical analyzing components, etc. Some integrated circuits could be also coupled with

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microfluidic parts like it is shown Figure 2.4 , an integrated circuit (IC)/microfluidic hybrid system for magnetic manipulation of biological cells [41]. This hybrid system consists of an IC and a microfluidic system fabricated on top. Biological cells attached to magnetic beads are suspended inside the microfluidic system that maintains biocompatibility. The IC contains a microcoil array circuit that produces spatially-patterned microscopic magnetic fields.

Figure 2.4. SiGe/Microfluidic Hybrid Prototype and Micrograph of the SiGe IC for controlling a microcoil array, [41]

- Tubing connection (for connecting supplier pump of chemical/biological substances and valve switch (internal or exterior valves)

- Motherboard: for connecting bottom substrate to control circuit

b. Advantages of microfluidics in chemical and biological applications:

In literature, a primary goal for much of the microfluidics community is to develop technologies that enhance the capabilities of investigations in biology and medical research. Many microfluidic studies describe methods that aim to replace traditional macro scale assays, and usually perform proof-of-concept experiments that attempt to demonstrate the efficiency of the new approach. A summary of the advantages are listed in Table 2-1.

Table 2-1. Some main advantages attained with microfluidic systems, [42] p-5

Microfluidic Advantage Description

Less sample and reagent

consumption Microfluidic devices typically require 10

2 – 103 less sample volume than

conventional assays.

Enhanced heat transfer Higher surface area-to-volume ratio of microfluidic channels increases effective thermal dissipation.

Faster separations Higher E-fields results in faster sample migration. Laminar flow Low Reynolds number flows reduce sample dispersion.

Electro-kinetic manipulation Electroosmotic flow enables fluid pumping with flat "plug-like" velocity profiles solely via applied E-fields.

Lower power consumption Fewer components and enhanced thermal dissipation require less power input.

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Parallelization Several assays can be “multiplexed”, or run in parallel on a single chip. Portability System integration and reduced power allows for assays to be

conducted using portable, hand-held device. Improved separation

efficiency Efficiency in electrophoretic and chromatographic separations (i.e. number of theoretical plates) proportional to L/d 2.2.2 Microfluidics’ theoretical aspects

In microfluidics, some problems appear with several factor: the size of the section of the channels, the nature of the surface (wettability), capillary force, high electric field, non-ideal fluid (blood, serum, etc.) and emergence of bubbles.

a.Reynolds number

Microfluidic devices almost always boast smooth laminar flow, as opposed to turbulent flow which is of a stochastic nature and marked by the presence of “eddies” that disrupt parallel streamlines. The Reynolds number (Re) compares the magnitudes of inertial force to viscous forces in a flow. It is a dimensionless parameter used to determine the transition from laminar to turbulent regimes (Re<2100-2400: laminar flow, no turbulence, Re>4000: turbulences).

The Reynolds number is defined as, [42]: D.u

Re

 (2_1)

where u is the flow velocity, ν is the kinematic viscosity (fluid dynamic viscosity on fluid density ratio), and D is the cylindrical channel diameter.

For a diameter of 10 to 100 µm in common conditions, Re is inferior to 1. The fluid is in laminar condition and we can see that the fluid behavior in microchannel can considerably deviate from those in macroscopic devices.

We can note that the surface forces, which originate due to intermolecular forces, could be important in microchannel flows. These forces are generally ignored at macro scale. Surface effects also alter the value of viscosity. It is found that the apparent viscosity is lower in the narrower channel, which is contrary to the expected trend [43, 44].

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b. The profile of velocity flow in a rectangle channel

The processes of the fabrication of the channels induce that the channel are never cylindrical. Figure 2.5 shows some plots of the contours of the velocity field and of the velocity field along the symmetry axes, [42]. The fact that, no analytical solution is known to the Poiseuille-flow problem with a rectangular cross-section, it always contains the side-wall effects.

Figure 2.5. (a) Contour lines for the velocity field νx(y,z) for the Poiseuille-flow problem in a rectangular

channel. The contour lines are shown in steps of 10% of the maximal value νx(0,h/2). (b) A plot of νx(y,h/2)

along the long centerline parallel to ey (unit vector on y-axis). (c) A plot of νx(0,z) along the short centerline

parallel to ez (unit vector on z-axis). (d) Geometry and shape of rectangle channel, [42].

In this case, the constant Reynolds number calculus, based on hydraulic diameter needs to introduce a quantity named “laminar equivalent” diameter [45] Dh defined

by: 2 h hw D h w   (2_2) h x h D .u Re   (2_3)

where ux is the average velocity along x axis

Based on the Re value and velocity profile of the liquid in channel, the mixing and cleaning process inside channel need to be controlled by controlling the flow rate of liquids in experiments in order to limit the side-wall effect. Designed specific mixing three zones are added in channel networks to improve the mixing of substances in experiments.

2.3 Magnetic bead-based immunoassay

This part of chapter will discuss about the state of the art of objectives in bead-based microfluidic systems applied for immunoassay.

x z y h w L (d)

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Nano/micro beads are frequently used as an immobilization/carrier surface to capture target analytes of interest in immunoassay, such as proteins and nucleic acids, from a biological sample. Nano/micro magnetic beads offer several advantages over planar surfaces (in standard ELISA protocol in wells) such as large specific surface to support biological interactions (increasing sensitivity or decreasing the limitation of analyte concentration). In the ELISA test, the typical sandwich structure is created as shown Figure 2.6. With the availability of the serial of commercial beads and multi-functionalization of microfluidic system, multiple targets in samples can be detected simultaneously.

The integration of microcoils as electromagnet into microfluidic systems for magnetic beads-based biosensing is interesting to replace the permanent magnet in ELISA test in micro test tube because in this case, the external magnetic field can be controlled automatically by an electric circuit to attract the beads, stop the trapping or maintain the magnetic field.

Figure 2.6. Sandwich structure of ELISA test on well’s platform and on beads’ surface

2.3.1 Magnetic micro/nano beads in immunoassay

In the development of building the bead-based immunoassay inside microfluidic chip, all materials such as: nano/micro magnetic beads with functional groups, primary antibody (or capture antibody) targets, second antibody with fluorescent label (or detection antibody), which concern to the immobilization antibody (IgG-type) in chapter of the application of microfluidic chip, are presented in the next section.

a.Introduction of functionalized magnetic beads

Specific antibodies are immobilized on beads’ surface and are enable to couple the specific target cells/proteins or viruses. Then, these beads are separated in the

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solution by using a magnetic force (generally a magnet). Currently, commercial micro/nano magnetic beads are available and they are used in research, pilot research or analytical devices [46, 47].

In current applications, beads can be classified into two main types: non-magnetic (silica and polymer beads) and magnetic/super-paramagnetic beads (iron oxide – Fe2O3 or Fe3O4, alloy or rare earth materials – NbFeB or SmCo), but the majority of these particles are super-paramagnetic, i.e. they have no magnetic memory. The size of these beads, which can play an important role, is in range of few nanometers to few micrometers and selects exactly the diameter.

The surface’s beads could be also functionalized/immobilized by specific functional groups corresponding to the aims of analyzing processes. There are some common types of functionalization, Figure 2.7:

Figure 2.7. Description of functional groups on beads’ surface

Many types of commercial super-paramagnetic nanoparticles (fabrication, physical and chemical properties, bio-compatible characterization, etc.) for many applications are introduced in [47]. In our study, we use commercial carboxyl magnetic beads of 300 nm diameter (Carboxyl-Adembeads, purchased from Ademtech).

These beads are uniform Super-Paramagnetic Iron Oxide Nanoparticles (SPIONs), Figure 2.8 shows the core shell structure of carboxyl-Adembeads.

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Physical characteristics (1):

- Diameter: 300 nm (CV max 20%) - Density: approx. 2.0 g/cm3

- Magnetization at saturation: approx. 40 emu/g - Specific surface area: 15 m2/g

- Iron oxide content: approx. 70% - COOH density: > 350 μmol/g - Solid content: 30 mg/ml (3%)

b. Magnetic properties of the materials of bead’s core

Generally, super-paramagnetic materials are preferred to fabricate cores of magnetic beads. The most important behavior of this material shares with para-magnetism the absence of magnetization when the external magnetic field is removed and with ferromagnetism the high levels of magnetization reached under the influence of a low magnetic field.

Other interesting properties these beads are their short relaxation time in which magnetization goes to zero after the effect of an external magnetic field (typical values are around 10-9-10-10s). Under an alternating external magnetic field, their magnetic moment is quickly reoriented.

A comparison of magnetic properties between ferromagnetic and super-paramagnetic materials is shown in Figure 2.9, we can see that the super-super-paramagnetic nanoparticles show no remanent magnetization (Mr).

Ferromagnetic material

Super-paramagnetic material

(a) (b)

Figure 2.9. (a) The magnetic moments of both ferromagnetic and superparamagnetic nanoparticles under an external magnetic field/ no magnetic field; (b) Typical magnetization curves of ferromagnetic (black line) and

super-paramagnetic (blue line) particles. [46, 48].

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15| P a g e 2.3.2 Immunoassay principles

2.3.2.1 Introduction of immunoassay

It is known that, an immunoassay is a test that relies on biochemistry to measure the presence and/or concentration of an analyte. There are two strategies using this method: qualitative (yes or no) or quantitative results.

Analytes can be normally large proteins, antibodies produced in human bodies as a result of an infection. These assays are highly adaptable and can be applied to many formats depending on the needs of the end user. Principle components of an immunoassay designed to detect a specific analyte, (such as influenza nucleoprotein, Alzheimer or cancerous markers) are the antibodies carefully selected to ensure the detection of the analyte at low concentration with high specificity, meaning will not react with similar antigens. The second feature of an immunoassay is the system that is designed to detect the binding of the specific antibody to the target analyte. Originally the signal from an immunoassay resulted from an enzyme acting on a substrate to yield a colored solution with the amount of color in the solution being equivalent to the amount of antigen in the test solution (ELISA).

ELISAs can be developed with a number of modifications to the basic procedure. The key step of ELISA format, immobilization of the antigen of interest, can be accomplished by direct adsorption to the assay plate/special surface (bead’s surface or channel’s surface) or indirectly via a capture antibody that has been attached to the surface. The antigen is then detected either directly (labeled primary antibody) or indirectly (labeled secondary antibody). The most powerful ELISA assay format is the sandwich assay. This type of capture assay is called a “sandwich” assay because the analyte to be measured is bound between two primary antibodies – the capture antibody and the detection antibody. The sandwich format is used because it is sensitive and robust, and we have already talked about this in the previous section.

Generally, the immunoassay can be classified into some approach methods:

- Competitive vs. non-competitive: In a competitive immunoassay there is a competition between labeled and unlabeled antigen for a limited number of binding sites on the antibody. All reactants are mixed together either simultaneously or sequentially. In a typical non-competitive assay (immunometric assay) an excess amount of an antibody is used to capture the analyte from the sample. A labeled second antibody

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is added and binds to the first antibody-antigen complex to form a sandwich. The complex formed is then measured. Non-competitive techniques are more sensitive and reproducible than competitive ones.

- Homogenous vs. Heterogeneous: Heterogeneous assays involve the binding of antigen to antibody followed by physical separation of the bound antigen from the unbound antigen prior to measurement. If the immunoassay does not require any physical separation step after mixing antigen and antibody and measurement of bound label occurs in the presence of unbound label, the assay is homogenous. Heterogeneous assays tend to be more versatile and sensitive.

- Isotopic vs. non-isotopic: Isotopic immunoassays use radioactive labels on the antigen or antibody. Non-isotopic methods use enzyme reactions, chromophores, fluorescent labels, or luminescent labels in order to detect the antigen-antibody complex.

- Limited reagent vs. excess reagent: In limited reagent assays, the number of available binding sites on the antibody is limited compared to the concentration of labeled and unlabeled antigen (a competitive immunoassay). Non-competitive assays are reagent excess assays.

In the study on the application of microfluidic chip for immobilizing antibody IgG-type on surface of the carboxyl-beads in next chapter, the process of immobilization is based on the method of competitive vs. non-competitive. This method is popularly used in most of ELISA test inside channel or in micro test tube. This method has also some advantages compares to other methods: high specificity, since two antibodies are used the antigen/analyte is specifically captured and detected;

suitable for complex samples, since the antigen does not require purification prior to measurement; flexibility and sensitivity, since both direct and indirect detection

methods can be used.

2.3.2.2Bio-components in immunoassay

As described in previous part, the sandwich ELISA format is preferred in many protocol of immunoassay (i.e. detection Alzheimer marker, cancerous marker, etc.) [49, 50]. In each ELISA assay, there are some basic components that are used in procedure. Three necessary ELISA reagents include: immune-sorbents (solid phase); conjugates (antibodies, antigens and enzyme); and substrates (substrates of enzyme).

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a. Capture antibody and immuno-sorbent/solid phase

All ELISAs rely on the specific interaction between an epitope, a small linear or three dimensional sequence of amino acids found on an antigen, and a matching antibody binding site. The antibodies used can be either monoclonal (derived from unique antibody producing cells called hybridomas and capable of specific binding to a single unique epitope) or polyclonal (a pool of antibodies purified from animal sera that are capable of binding to multiple epitopes). However, polyclonal antibodies are more typically used for the secondary detection layer in indirect ELISAs, while monoclonal antibodies are more typically used for capture or primary detection of the antigen.

In sandwich ELISA structure, primary antibodies (conjugate antibodies) supplied as commercial products are immobilized on solid surface. In some cases, the immune-sorbent is only coated by functional groups (amine or carboxyl groups), antibodies will be immobilized on activated solid surface in separating step:

The Immuno-sorbent (Solid support which has been coated antibodies or antigens) is commonly provided as commercial product (i.e. wells array or particles with nano or micro size of diameter). For particles, they were introduced in previous part. For wells array, they are usually made in polystyrene, cheap material which can present large kind of shapes. This material is strong in adsorbing protein. Antibody or protein antigen remains activity after adsorbed on it.

The Primary antibodies (or conjugated antibodies) are immobilized on solid supports. These antibodies are the specific ones which able to conjugate with a specific targets in sample. Thus, proteins G, A, D, E or M type are usually used. In many application, IgGs (main antibody in blood Figure 2.10) are commonly used as a primary antibodies, because of the simple manipulation, population and cost. It is the only isotype that can pass through the placenta, and transferred from the mother's body protects a newborn until a week after birth.

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Figure 2.10. Population of 5 types of antibodies in blood (%) (1)

 Chemical and biological characterization of antibody IgG-type:

In the chapter 4, this antibody will be used for all experiment of immobilization antibody on carboxyl magnetic beads. All properties as shown below are important to understand the interaction of this antibody to other substances in reaction solution.

It is known that, immunoglobulins (Igs) are produced by B lymphocytes and secreted into plasma. The Ig molecule in monomeric form is a glycoprotein with a molecular weight of approximately 150 kDa that is shaped more or less like a Y [51]. Basic structure of the Ig monomer (Figure 2.11) consists of two identical halves connected by two disulfide bonds. Each half is made up of a heavy chain of approximately 50 kDa and a light chain of approximately 25 kDa, joined together by a disulfide bond near the carboxyl terminus of the light chain. The heavy chain is divided into an Fc portion, which is at the carboxyl terminal (the base of the Y), and a Fab portion, which is at the amino terminal (the arm of the Y). Carbohydrate chains are attached to the Fc portion of the molecule. The Fc portion of the Ig molecule is composed only of heavy chains. Fc regions of IgG and IgM can bind to receptors on the surface of immune-modulatory cells such as macrophages and stimulate the release of cytokines that regulate the immune response. The Fc region contains protein sequences common to all Igs as well as determinants unique to the individual classes. These regions are referred to as the constant regions because they do not vary significantly among different Ig molecules within the same class.

Each IgG monomer is capable of binding two antigen molecules at each amino terminal of the left and right Y’s arm. However, amine groups at these positions are different properties and have different behavior in solution during reaction time [52].

(1) http://www.kyowa-kirin.com

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Figure 2.11. Antibody (IgG) 2D and 3D structures (1, 2, 3)

In generally, the immobilization of IgG does not discriminate between possible attachment points near or removed from the specific binding site, which results in spatial orientation of antibodies on the supports that might prohibit formation of an antibody-antigen complex. For instance, if multiple lysine groups, Figure 2.11.b, are present on the surface of an antibody molecule, multiple attachments might occur. This may result in different orientations of the antibody on the support surface depending on which lysine group binds to the support. When immobilization occurs through the antigen binding sites on the Fab’ portions, the ability of that antibody to bind antigen may be severely impaired or eliminated entirely [53].

b. Detection antibodies (antibodies conjugated enzyme)

Detection antibodies, as commonly called secondary antibodies, are specific anti-Fabs of antibodies. A secondary antibody aids in the detection, sorting or purification of target antigens by binding to a primary antibody, which directly binds to the target antigen. Secondary antibodies offer increased sensitivity through the signal amplification that occurs at two labeled secondary antibodies binding to the two Fab fragments of primary antibodies. In addition, a given secondary antibody can be used with any primary antibody of the same isotype and target species, making it a more versatile reagent than individually labeled primary antibodies.

(1) http://www.sigmaaldrich.com (2) http://visual-science.com (3) http://www.piercenet.com/method/antibody-labeling-immobilization-sites (b) (a) NH2 COOH COOH NH2 NH2 NH2 Antigen binding Antigen binding Pepsin cleavage F(ab’)2 fragment Fab fragment papain cleavage Fc fragment Isotype determinants μ, γ, α, δ, or ε

Heavy chain constant region Heavy chain variable region Light chain variable region Light chain variable region Carbohydrate Antigen binding site Disulfide bonds Papain cleavage site Papain cleavage site

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Second antibodies are commonly conjugated with enzyme, or some time with only fluorescence/dye. They may be provided in several formats: whole IgG, divalent F(ab')2 fragments and monovalent Fab fragments. Figure 2.12 shows the structure of FITC-conjugated antibody (1) (type G) that is commonly used in immunoassay. This substance is also used to detect the grafted antibody IgGs on carboxyl beads in the study of chapter 4.

Figure 2.12. FITC-conjugated antibody (IgG)

c. Enzyme and antigens

- Enzymes used in ELISA should meet requirements such as high purity, high conversion rate, favorable specificity, stable properties, rich resources, cheap price and remaining active component and catalytic capacity after becoming conjugate.. In addition, corresponding substrate is easy to be made and stored. Non-ferrous products are easy to be detected. In ELISA, horseradish peroxidase (HRP) and alkaline phosphatases (AP) are usually used.

- Antigen: Antigens (targets in analytes) are components which are interested in analyzing propose. Antigens are any substance that stimulates the immune system to produce antibodies. Antigens can be bacteria, viruses, or fungi that cause infection and disease. They can also be substances, called allergens, which bring on an allergic reaction. Blood transfusions containing antigens incompatible with those in the body's own blood will stimulate the production of antibodies, which can cause serious, potentially life-threatening reactions.

2.3.3 General protocol of magnetic bead-based immunoassay

We have already mentioned that, the bead-based immunoassay present a very high sensitivity and selectivity [6, 54-56].

(1) Fluorescein isothiocyanate – conjugated antibody

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In bead-based immunoassay, the reacted or immobilized magnetic beads can be easily separated from the reaction mixtures with a magnet and re-dispersed immediately following removal of the magnet. Therefore, this protocol has been used in a variety of research fields, such as food safety [57], environment monitoring [58], and clinical diagnosis [59].

Figure 2.13 shows a general protocol of bead-based immunoassay that is performed in micro test tube based on immune-magnetic capture and bacterial intrinsic peroxidase activity for a quick detection of Shewanella oneidensis (1), [60]. This protocol includes two processes: (a) The preparation of functionalized magnetic beads (MBs) and immobilizing primary antibodies on beads’ surface; (b) The procedure of the colorimetric immune-magnetic assay.

Figure 2.13. Schematic for the antibody/MBs preparation and colorimetric immune-magnetic assay.(a) Preparation procedure of antibody/MBs. (b) The procedure of the colorimetric immune-magnetic assay [60].

In general, the protocol of basic bead-based immunoassay includes following step: - Activating the functionalized nano/micro magnetic beads by activation chemicals –

chemical process (e.g. for carboxyl magnetic beads, EDC and Sulfo-NHS (2) are used to activate the carboxyl groups).

- Immobilizing primary/capture antibody (e.g. IgG-type) on activated beads’ surface and blocking immobilized antibodies by bovine serum albumin (BSA) – blocking agent.

- Capturing antigens or biomarker in sample solution, as called immuno-magnetic capture. In this step, antigens/biomarkers are captured by primary antibodies

(1) A bacterium which can reduce poisonous heavy metal and can live in both environments with or without oxygen (2) EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; Sulfo-NHS: N-hydroxysulfosuccinimide

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- Detecting antigens by fluorescent detection or other methods (e.g. electrochemical detection, time-resolved fluorescence, enzyme-based photometric etc.)

In the study of chapter 4, the immobilization of antibody type-G will be based on this protocol.

2.4 Microfluidics in magnetic bead-based immunoassay

Like the method in standard immunoassay, the microfluidic immunoassay is mainly performed basing on bead-based immunoassay. It means that, the antibodies and antigens can be immobilized on the surface of the microchannels or beads. Immobilization on surfaces of microchannels requires additional steps during the microfabrication process and may suffer from poor reproducibility and reliability. In addition, bead-based immobilization can be performed outside the microchip [61, 62].

2.4.1 Magnetism and trapping/transporting magnetic beads in microfluidics

Manipulating magnetic micro/nano beads in microfluidic chips can be done efficiently with permanent/electro magnets [13, 63-65]. The permanent magnet is known to generate a strong magnetic flux density, i.e. from few hundreds mT to 1 T [16, 66, 67]. However, the permanent magnet only generates a permanent field. Consequently, removing or diminishing the magnetic field requires mechanically moving the magnet. Therefore, the magnetic field cannot be turned off immediately.

2.4.1.1Permanent magnet for manipulating magnetic beads in microfluidics

The best magnetic material using to fabricate permanent magnet is neodymium iron boron (NdFeB). It can generate very strong and stable magnetic field event in small dimensions.

Figure 2.14 shows the magnetic field simulation of different shapes of NbFeB magnets, which generate complex magnetic field patterns event on the small size of magnets. The Figure 2.14 (a) shows the homogenous magnetic field generating by a magnet bar, this field is required for NMR spectroscopy and magnetohydro-dynamic pumping. But it is also suitable for trapping stably magnetic beads in bead-based immunoassay. Figure 2.14 (b) and (c) show inhomogeneous magnetic field generating by needle magnet in NdFeB material or layered structures magnet in iron with the aim is to trap particles or transport materials within a fluid volume.

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Figure 2.14. Magnetic fields from permanent NdFeB magnets modelled with FEMM-software [68]: (a) A homogeneous field at a distance of 1 mm along the surface of a large magnet. (b) An inhomogeneous field

100mm above the surface of a tapered magnet. (c) A magnetic field with local minima and maxima at 100mm distance above a stack of alternating iron and aluminum blocks.

As an example of use of external magnet, B.A. Otieno et al., 2014 [30], describe the incorporation of a novel on-line chamber to capture cancer biomarker proteins on magnetic beads, which are derivatized with 300,000 enzyme labels and 40,000 antibodies into a modular microfluidic immunoarray ( Figure 2.15).

Figure 2.15. Photographs of microfluidic system for on-line protein capture and detection using magnetic beads: (A) Capture chamber in which target proteins are captured on-line from the sample by heavily labeled

HRP-antibody-magnetic beads to form protein–bead bio-conjugates. These are washed, and then flowed into the detection chamber (B) in the modular microfluidic system (C). The magnet (D) traps bio-conjugate

beads in the channel during injection of sample and washing, and is removed for transfer of beads to the detection chamber, [30].

The capture chamber features an internal layer of flexible PDMS prepared on a machined template to have an oval hole sandwiched in between two flat, machined PMMA plates (Figure 2.15. A). When bolted tightly together, this assembly forms an

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oval cylindrical channel 1.5 mm wide, 1.8 mm thick and 100 ± 2 μL in volume housing a tiny magnetic stir bar. In the capture chamber, the magnetic beads are mixed and trapped by the magnetic stir bar. This magnetic bead position is controlled magnetically in the on-line capture chamber for rapid binding of proteins, washing, and delivery to the detection chamber.

2.4.1.2Electromagnet for manipulating magnetic beads in microfluidics

The use of electromagnets can be a very attractive alternative solution because these magnetic field sources can be controlled and have a fast response. Electromagnets are coils with or without an associated magnetic circuit (e.g., magnetic core). However, electromagnets generate a weaker magnetic field than the one generated from permanent magnets for equivalent dimensions. To enhance the magnetic field strength, high current or special design with extra parts is required [69]. In general, the design of electromagnet is based on some types of geometries such as planar/spiral coils, 3D coils or magnetic tweezers, coil with encapsulated magnetic material cores (e.g. ferromagnetic oxide, Permalloy) [69-72]. Single coil or coils array have been already integrated in microfluidic chip for trapping, mixing or moving magnetic beads [70, 73, 74].

In the case of planar coils (fabricated in single layer coil or multi-layer coil in order to improve the magnetic flux density [41, 70, 75]), the most popular shapes of electromagnet, the magnetic flux density is usually low, from few mT to few hundreds mT depending on the parameters of the coils [76, 77].

Of course, the magnetic field depends on the geometry of coils, and it also depends on the current density applied to coils. When designing coils, in addition to generating the maximum magnetic flux density or magnetic force, the power consumption/power efficiency and heat generation of coils are careful trade off as well.

To design microcoils for application of trapping/mixing magnetic beads and integration into microfluidic chip, the simulation and calculation of magnetic field or magnetic force are required. The results of the calculation based on a simulation model will definite the geometry of coils or other parameters such as: number of turns, dimensions of conductor, etc. The optimal geometries of coils are based commonly on general requirement of the maximum value of magnetic field/force, the

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minimum value of power consumption or homogeneous magnetic field, etc. The magnetic field of planar coil is usually calculated basing on the Finite Element Modelling/Method (FEM), and the simulation/calculation software is commonly used as a useful tool for the big simulation (i.e. MatLab®, ANSYS®, COMSOL Multiphysics®).

Figure 2.16. Wire arrangement for modeling a planar coil. (a) Wire arrangement of a planar rectangular coil. (b) Arrangement of a horizontal wire segment with finite length. (c) Coordinate transformation to formulate

the magnetic field of a vertical segment of wire [76].

Beyzavi et al. [76] used the simple analytical model for the magnetic field of a single straight wire to a spiral shaped rectangular planar coil. This model can then be used for the calculation of the strength, the flux and the resultant force of the magnetic field generated by the microcoil. It also helps to find maximize the field strength and the force. In Figure 2.16, a model of planar coil is used to calculate magnetic field and force. The planar coil is composed of a series of current-carrying wires with finite length, and the magnetic field is calculated from each magnetic field element created by each wire of a finite length should be obtained in the calculation domain. Figure 2.16.a shows the wire arrangement of a planar rectangular coil. Each straight segment of the coil is termed here as a wire segment. Four consecutive wire segments form a turn. Thus, if the number of segments is n, the number of turns is approximately n/4.The superposition of the magnetic fields of all these segments results in the total magnetic field of the coil.

Figure 2.17 shows the typical results of the model for a rectangular coil. The field strength was calculated for a line running along the x-axis and through the center of the coil. The modeling results show that the total magnetic field (Htotal) of a planar

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coil has almost the same trend and magnitude as the z-component of its magnetic field strength (Hz).

Figure 2.17. Modeling results for magnetic field distribution of a single planar coil on the central line of the coil (n=47, g=200 µm, I=0.3A, y=0 mm, z=1 mm) [76]

R. Fulcrand et al. [70] designed an integrated complex magneto-fluidic lab-on-chip, which is composed of multiple inlets and multiple microcoils within one single device. This simulation model is more complicated than model shown above. A serial of microcoils designs are proposed, and the magnetic field and magnetic force for the trapping and driving beads in microfluidic chip are calculated. The simulation models and results are shown in Table 2-2 and Table 2-3.

Table 2-2. Micro-coils designs and dimensions [70]

Table 2-3. Maximum magnetic flux density, field gradient and magnetic force, 5 μm and 50 μm into the microfluidic channel for each design. I = 100 mA [70].

As shown in Table 2-3, the magnetic flux density along the z and x axes Bx

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values Bz,max obtained with square and circular for z = 5 µm, i.e. at the bottom of the channel, are approximatively 3 times higher than those obtained with serpentine structures. Figure 2.18 shows the results of trapping and driving magnetic beads inside microfluidic chip with the arrangement of microcoils in channel.

Figure 2.18. Video sequence showing the manipulation of microbeads batch by successive trapping and release. (a) Flow is established and microbeads are injected in the main channel, all micro-coils are switched

off. (b) Micro-coil No. 1 is supplied with a current I = 70 mA, microbeads are trapped. (c) Micro-coil No. 1 is switched off while micro-coil No. 2 is simultaneously powered at I = 70 mA, the beads batch is shifted from micro-coil 1 to micro-coil 2. (d) The bead batch is immobilized on micro-coil 2 as long as necessary and

released in the same way than previously (e) and (f), [70].

A 0.75 nL/s laminar flow was established and microbeads were injected. The first micro-coil was then supplied with 70 mA during 5 s in order to trap a batch of microbeads (Figure 2.18 (b)), which is subsequently displaced by the sequential actuation of two other micro-coils (Figure 2.18 (c–f). Microbeads’ batches can eventually be deflected into a desired outlet taking advantage of the uni-directionality of laminar flows.

One important disadvantage of the use of electromagnets is heat generation induced by the Joule effect [74, 78]. Evaluating the temperature raise is of crucial importance for chips using biological species: an operating temperature range is often required for biological binding reactions or temperature needs to be kept below certain limits in order not to destroy the biological species. In the literature, temperature raise originated from the coils is compensated by several means. In [79], a cooling channel network is integrated in the multi-layered thermal chip to extract heat from coils. In [41], the IC/microfluidic hybrid chip with microcoil array is assembled with a Peltier module (thermoelectric cooler) in order to keep the system temperature at 37°C. A good strategy to limit the need of cooling devices consists in limiting the thermal emission of the coil. Obtaining magnetic field with low energy

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